Biochemistry 1991, 30,4594-4599
4594
Gouaux, J. E., Krause, K. L., & Lipscomb, W. N. (1987) Biochem. Biophys. Res. Commun. 142,893-897. Hermes, J. D., Roeske, C. A,, O'Leary, M. H., & Cleland, W. W. (1982)Biochemistry 21, 5106-5114. Huskey, W. P. (1991) in Origins and Interpretations of Heavy-Atom Isotope Effects (Cook, P. F., Ed.) Chapter 2, CRC Press, Boca Raton, FL. Kinoshita, Y., Ruble, J. R., & Jeffrey, G. A. (1981)Carbohydr. Res. 92, 1-7. Kraut, J., & Jensen, L. H.(1963)Acta Crystallogr. 16,79-88. LaReau, R. D., Wan, W., & Anderson, V. (1989)Biochemistry 28, 3619-3624. Leung, H. B., & Schramm, V. L. (1981)J . Biol. Chem. 256, 12823-1 2829. Markham, G.D.,Parkin, D. W., Mentch, F., & Schramm, V. L. (1987)J . Biol. Chem. 262, 5609-5615. Melander, L., & Saunders, W. H.,Jr. (1980)Reaction Rates of Isotope Molecules, pp 239-246,Wiley-Interscience, John Wiley and Sons, New York. Mentch, F., Parkin, D. W., & Schramm, V. L. (1987)Biochemistry 26,921-930. Northrop, D. B. (1981)Annu. Rev. Biochem. 50, 103-131. Parkin, D. W., & Schramm, V. L. (1984)J . Biol. Chem. 259, 941 8-9425. Parkin, D. W., & Schramm, V. L. (1987)Biochemistry 26, 9 13-920.
Parkin, D.W., Leung, H. B., & Schramm, V. L. (1984)J . Biol. Chem. 259,941 1-9417. Rodgers, J., Femec, D. A,, & Schowen, R. L. (1982)J . Am. Chem. SOC.104, 3263-3268. Scharschmidt, M., Fisher, M. A., & Cleland, W. W. (1984) Biochemistry 23, 547 1-5478. Schramm, V. L. (1974)Anal. Biochem. 57, 377-382. Schuber, F., Travo, P., & Pascal, M. (1976)Eur. J . Biochem. 69,593-602. Sims, L. B., & Lewis, D. E. (1984) Zsot. Org. Chem. 6, 161-259. Sims, L. B., Burton, G. W., & Lewis, D. E. (1977)Quantum Chemistry Program Exchange No. 337,Indiana University, Bloomington, IN. Sinnott, M. L. (1987)in Enzyme Mechanisms (Page, M. I., & Williams, A., Eds.) pp 259-297, Royal Society of Chemists, Burlington House, London. Taylor, R., & Kennard, 0. (1982)J . Am. Chem. SOC.104, 3209-3212. Wilson, E. B., Decius, J. C., & Cross, P. C. (1955)Molecular Vibration, McGraw-Hill Book Company, Inc., New York. Wi6rkiewicz-Kuczera, J., & Karplus, M. (1990)J. Am. Chem. SOC.11 2, 5324-5340. Zoltewicz, J. S . , Clark, F. D., Sharpless, T. W., & Grabe, G. (1970)J . Am. Chem. SOC.92, 1741-1750.
Structural and Functional Roles of Cysteine Residues of Bacillus polymyxa @-Amylase Nobuyuki Uozumi, Tsukasa Matsuda, Norihiro Tsukagoshi,* and Shigezo Udaka Department of Food Science and Technology, Faculty of Agriculture, Nagoya University, Chikusa- ku, Nagoya 464-01,Japan Received October 3, 1990; Revised Manuscript Received January 2, 1991
ABSTRACT: Bacillus polymyxa @-amylasecontains three cysteine residues at positions
83,91,and 323,which can react with sulfhydryl reagents. To determine the role of cysteine residues in the catalytic reaction, cysteine residues were mutated to construct four mutant enzymes, C83S,C91V, C323S,and C-free. Wild-type and mutant forms of the enzyme were expressed in, and purified to homogeneity from, Bacillus subtilis. A disulfide bond between Cysg3and Cysg' was identified by isolation of tryptic peptides bearing a fluorescent label, IAEDANS, from wild-type and C91V enzymes followed by amino acid sequencing. Therefore, only Cys323contains a free SH group. Replacement of cysteine residues with serine or valine residues resulted in a significant decrease in the k,,/K, value of the enzyme. C323S,containing no free SH group, however, retained a high specific activity, approximately 20% of the wild-type enzyme. None of the cysteine residues participate directly in the catalytic reaction.
%e enzyme @-amylase(a-1,4-glucan maltohydrolase, EC 3.2.1.2)catalyzes the liberation of @-anomericmaltose from the nonreducing ends of a-1,4-glucan and is present in certain bacteria (Marshall, 1974;Murao et al., 1979;Shinke et al., 1974;Takasaki, 1976;Hyum & Zeikus, 1985) as well as in higher plants (Bernfeld, 1955;Kreis et al., 1987). Five genes encoding @-amylasehave been cloned and sequenced from both prokaryotes and eukaryotes (Kawazu et al., 1987;Rhodes et al., 1987;Kitamoto et al., 1988;Toda et al., 1988;Kreis et al., 1987;Mikami et al., 1988). Three highly conserved se-
* To whom correspondence should be addressed.
quences are recognized among them and are suggested to comprise the active site (Mikami et al., 1988). @-Amylases characterized to date are sensitive to various sulfhydrylmodifying reagents and are considered to contain an S H group essential for the activity (Murao et al., 1979;Hyum & Zeikus, 1985;Gertler & Birk, 1966;Spradlin & Thomas, 1970;Higashihara & Okada, 1974;Uehara & Mannen, 1979). The exact role of cysteinyl residues in the enzymatic action, however, remains uncertain, since the derivatization of the S H groups with alkylating reagents might inhibit the catalytic action only by steric hindrance at the active site (Mikami et al., 1980).
0006-2960/91/0430-4594$02.50/00 199 1 American Chemical Society
Bacillus polymyxa @-Amylase 0
I
1
Biochemistry, Vol. 30, No. 18, 1991 4595 2
3
I Kb
pYN520
B
hl
p Y N 5 4 1
TTG
FIGURE 1 :
A
T A A
Diagrammatic presentation of the @-amylasegene from
B. polymyxa. The upper half represents the correlation of the 8and a-amylase coding regions on plasmid pYN520, while the lower half shows the amylase gene on plasmid pYN541 used in this study.
Closed triangles indicate the positions of cysteine residues. The start codon (TTG) and stop codon (TAA) are also indicated. The abbreviations for restriction enzyme sites are B, BamHI; E47, Eco47III; ET, EcoT221; EV, EcoRV; Hc, HincII; and K, KpnI. Bacillus polymyxa produces multiform @-amylaseswith approximate molecular masses of 70, 56, and 42 kDa and a 48-kDa a-amylase (Kawazu et al., 1987). The amylase gene contains in-phase 0-and a-amylase coding sequences in the 5’ and 3’ regions, respectively. A precursor protein, a 130-kDa amylase, has both @- and a-amylase activities and is proteolytically cleaved to produce multiform 0-amylases and a 48kDa a-amylase after secretion (Uozumi et al., 1989). The three major @-amylasescontain three cysteine residues and have essentially the same enzymatic properties (Kawazu et al., 1987). To determine whether the E. polymyxa @-amylasecontains an essential active cysteine residue, we employed site-directed mutagenesis to replace three cysteine residues by serine or valine residues utilizing the DNA fragment encoding the 42-kDa 0-amylase. The mutant enzymes were purified and characterized. The E . polymyxa @-amylasecontained a disulfide linkage. None of the cysteine residues appear to be essential for catalytic activity. MATERIALS AND METHODS Bacterial Strains, Media, and Transformation. L broth (Miller, 1972) and antibiotic medium 3 (Difco) were used to grow Escherichia coli JM103 (Maniatis et al., 1982) and Bacillus subtilis 1A289 (amyE sacA321 aroI906 metB5; Bacillus Genetic Stock Center, The Ohio State University, Columbus), respectively. When required, ampicillin and kanamycin were added at 50 and 10 pg/mL, respectively. The transformation of E . coli and E . subtilis was carried out as described previously (Kawazu et al., 1987; Uozumi et al., 1989). 0-Amylase-producing transformants were identified by staining the plates with a 1.7 mM 12-KI solution (Tsukagoshi et al., 1984). Plasmids. pYN520 containing in-phase @- and a-amylase coding sequences of the E . polymyxa amylase gene was described previously (Uozumi et al., 1989). pYN4941 was constructed as follows: a 2.4-kb BamHI-KpnI fragment containlng the @-amylase coding region was isolated from pYN520 and inserted between the BamHI and KpnI sites on pUCl18 (Figure 1). A universal translation terminator, S-GCTTAATTAATTAAGC-3/ (Pharmacia Fine Chemicals, Piscataway, NJ), was inserted into the EcoRV site after removal of the 0.3-kb EcoRV fragment on the resultant plasmid. pYN541 was constructed as follows: a 2.1-kb BamHI-EcoRI fragment was isolated from pYN4941 by use of the EcoRI site located 12 bp downstream of the 3’ end of the insert and inserted between the BamHI and EcoRI sites on PUB1 10, followed by the transformation of E . subtilis 1A289 to kanamycin resistance. Site-Directed Mutagenesis of the @-AmylaseGene. The 0.6-kb Eco47III-EcoT221 and 0.4-kb KpnI-HincII fragments
on pYN4941 containing regions I and I11 conserved among @-amylases, respectively, were inserted between the corresponding sites on M13mp19. Single-stranded M13mp19 DNA containing the insert was subjected to mutagenesis with the aid of the oligonucleotide-directed in vitro mutagenesis system (Amersham Corp.). To confirm that only the desired mutations occurred during the manipulations, the gene from each was sequenced for its entirety by the dideoxy chain-termination method (Sanger et al., 1977). The Eco47III-EcoT22I or KpnI-HincII fragment with the confirmed mutations was inserted between the corresponding sites on pYN4941 and used for the transformation of E . coli JM103 to ampicillin resistance. To analyze the gene products, the BamHI-EcoRI fragment containing the mutated gene was subcloned onto pUBllO as described above and expressed in B. subtilis. Mutagenic deoxyoligonucleotides 5‘-CAACGCATAAGAGTGGAGG-3’, 5’-GTAGGAGATGACGTCAACATCCC-3’, and 5’-GACATTTACTAGCCTGGAG-3’were synthesized at the Center for Gene Research of Nagoya University and were used to obtain pYN541 -C83S, pYN 541 -C9 1V , and pY N541 -C323S, respectively. pYN541-C-free was constructed as follows: the Eco47IIIEcoT22I fragment on pYN541 -C83S containing region I was further mutated at codon 91 to produce double mutations, C83S-C91V, followed by insertion in the corresponding region on pYN541-C323S. Purification of ,&Amylases. E . subtilis 1A289 cells with plasmids containing wild-type and mutant @-amylasegenes were grown for 40 h at 28 OC in antibiotic medium 3 supplemented with 0.5% soluble starch and 10 pg/mL kanamycin. @-Amylaseswere purified as described previously (Kawazu et al., 1987) with some modifications. The nonbinding fractions containing @-amylaseon a DEAE-cellulose column were pooled, dialyzed against 10 mM acetate buffer (pH 6.0), and applied to a column of CM-cellulose equilibrated with the same buffer. The nonbinding fractions containing the enzyme activity were pooled and dialyzed extensively against 100 mM phosphate buffer (pH 7.0) containing 25% ammonium sulfate, followed by fractionation on a phenyl-Sepharose CL-4B column previously equilibrated with the same buffer. The enzyme was eluted at a flow rate of 18 mL/h from the column with 10 mM phosphate buffer (pH 7.0). The 0-amylase-containing fractions were pooled and dialyzed against 50 mM phosphate buffer (pH 7.0). Protein Derivatization. The general conditions for protein labeling and isolation of resultant derivatives were as described previously (Sanger et al., 1977; Gorman et al., 1987). Wild-type and mutant C91V @-amylases(400 pg of each) were labeled at 25 OC for 2 h with 4.5 mM N-(iodoacetyl)-N’-@sulfo-1-naphthy1)ethylenediamine(IAEDANS)’ in 75 mM Tris-HC1 buffer (pH 8.0) containing 6 M guanidine hydrochloride and 4.5 mM EDTA. The derivatized proteins were protected from light to avoid photodecomposition of the fluorophore. This also applied to the analytical procedures described below. The reactions were stopped by adjustment of the pH of the reaction mixture to 5 with acetic acid, followed by extensive dialysis against 100 mM Tris-HCI buffer (pH K O ) , and then dialysis against the same buffer containing 1 mM CaCl,. Protein Cleauage and Peptide Separation. Protein derivatives were digested by the addition of aliquots of 1% (w/v) I Abbreviations: pCMB, p-chloromercuribenzoate; DTNB, 5,5’-dithiobis(2-nitrobenzoate); a-EPG, 2,3-epoxypropyl a-o-glucopyranoside; IAEDANS, N-(iodoacetyl)-N’-(8-sulfo-1-naphthyl)ethylenediamine, NEM, N-ethylmaleimide.
Uozumi et al.
4596 Biochemistry, Vol. 30, No. 18, 1991 I
I11 323
I1
83
91
A
A
A
B.polymysa
7 7 IISTHKCCGNVCDDCNIPLPSW 9 8
1 5 9 CPSCELRYPSYYP 1 7 2 3 1 9 LTFTCLEEISDS 3 2 3
C.thermosulfurogenes
7 7 I).ISTHACCGNVGDTVNIPIPSW 9 8
1 5 9 CPSCELRYPSYNP 1 7 2 3 2 0 ?ITFTCiEElDDS 3 3 0
Soybean
89
Barley
87
Sweet Potato
9 0 IMSFIIQCCCNVADAVI~IPIPQW1 1 1 1 8 4 CAACELRYPSY-P 1 3 5 3 4 1 LSFTC'LE?lRDS 3 5 1
COKSERVED
* *** ******* *** *** *** * ******* ****** * IMSFHQCGGNVCDIVNIPIPQW ************* ******** IMSFllQCGGNVCDAVNIPIPQW *********** *** ****** I-S-11-CCGNV- D - - -
I P-P-W
110 182 108 180
*********** * ** ******** * CPACELRYPSY- P ***** ***** * GPAGEHRYPSY-P * *** ***** * C--CE-RYPST-P
193 339
******* * * ****** ** LNFTCLEEIRDS 3 4 9 **** INI~TCAEHItDL 3 4 7 **** **** **.?*
131 337
- - FTC - E El - L)-
FIGURE2: Conserved amino acid sequences in three homologous regions (I, 11, and 111) of @-amylases. Identical residues are denoted by asterisks between the sequences. The amino acids are numbered from the N terminus of each mature enzyme. Closed triangles indicate positions of cysteine residues of the B. polymyxa P-amylase. Amino acid sequences of the following amylases were aligned to maximize the homologies: B. polymyxa (Kawazu et al., 1987; Rhodes et al., 1987; Uozumi et al., 1989), C. thermosulfurogenes (Kitamoto et al., 1988). soybean (Mikami et al., 1988), barley (Kreis et al., 1987), and sweet potato (Toda et al., 1988). Conserved amino acids among five @-amylasesare shown at the bottom.
trypsin at a ratio of @-amylaseto trypsin by weight of 50 to 1. After incubation at 37 "C for 16 h, digestion was terminated by freezing the reaction mixture and subsequent lyophilization. The resultant peptides were dissolved in 0.1% CF,COOH in H 2 0 and sepatated by HPLC (JASCO, Tokyo, Japan) on a reversed-phase column (Biofine RPC-PO, 4.6 X 150 mm; JASCO). Chromatography was performed at a flow rate of 1 mL/min with a linear gradient of increasing percentages of CH,CN relative to H 2 0 while a constant concentration of 0.1% (v/v) CF3COOH was maintained. Programs for rates and extents of increase in CH3CN were optimized for each particular separation. Absorbance of the eluates was monitored at 220 nm in addition to fluorescence emission monitored at 540 nm due to excitation at 340 nm. Peaks of fluorescence were collected manually and lyophilized. If necessary, samples were digested further with trypsin, and peptides bearing fluorescence were isolated as described above. Other Analytical Procedures. The NH2-terminal amino acid sequences were determined with a gas-phase sequence analyzer (AB1 477A-120A protein sequencer). The circular dichroism spectrum of each protein was measured at room temperature with a JASCO J-5OOC spectropolarimeter. The @-amylaseactivity was determined at 25 "C with soluble starch as a substrate (Kawazu et al., 1987). Protein was determined with bovine serum albumin as the standard (Lowry et al., 195 1). The purity of protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli, 1970).
RESULTS Construction and Expression of the Mutant @-Amylase Genes. The amino acid sequences deduced from various @amylase genes were optimally aligned to the B. polymyxa sequence. The alignment revealed three highly conserved regions (Figure 2). The B. polymyxa @-amylasecontained one additional cysteine residue in region I, which was replaced with a valine residue in the other enzymes. Taking into consideration the sequence similarity, we constructed four mutant genes by means of oligonucleotide-directed mutagenesis. The cysteine codons at 83 and 323 were replaced by a serine codon to construct C83S and C323S, respectively. C91V, with the cysteine codon at 91 replaced by a valine codon, contained the sequence most homologous to the other enzymes. In C-free, three cysteine codons were replaced by serine codons at codons 83 and 323 and by a valine codon at codon 9 1. The mutant enzymes were expressed in B. subtilis and purified by the same procedure used for the wild-type (Wt) enzyme. The proteins obtained were homogeneous as judged by SDS-polyacrylamide gel electrophoresis and had approximate molecular masses of 42 kDa (Figure 3).
KDa
- 94 - 67 - 43 - 30
- 20 1 2 3 4 5 6 FIGURE3: SDS-polyacrylamide gel electrophoretic profiles of the
purified @-amylases.The &amylases were purified from B. subtilis carrying pNY541 (lane l ) , pNY541-C83S (lane 2), pNY541-C91V (lane 3), pNY541-C323S (lane 4), or pNY541-C-free (lane 5). A 0.4-pg sample of each protein was analyzed. Molecular weight marker proteins (phosphorylase b, 94 000; bovine serum albumin, 67 000; ovalbumin, 43 000; carbonic anhydrase, 30 000; soybean trypsin inhibitor, 20 100; a-lactoalbumin, 14 400) were simultaneously electrophoresed (lane 6). The gel was stained with Coomassie brilliant blue R-250.
Identification of Disulfide Bond. To assess whether two cysteine residues at 83 and 91 in the B. polymyxa P-amylase form a disulfide bond, both Wt,and C91V enzymes were derivatized with a fluorescent label, IAEDANS, under denaturating conditions. Tryptic digestion of fluorescent derivatives followed by high-performance liquid chromatograhy yielded two and three major peptides bearing the fluorescent label from Wt and C91V proteins, respectively (Figure 4). Among those five peptides, peptide C91 V-3 was unique and should be generated as the consequence of mutation. Wt-1 and -2 were eluted from a Biofine RPC-PO column at exactly the same retention time as that of C91V-1 and -2, respectively. Upon further digestion of C91V-2 with trypsin, a major peptide, C91V-2', bearing the fluorescent label was eluted at nearly the same position as those of Wt-1 and C91V-1. All the major fluorescent peptides, in which cysteine residues should be derivatized with IAEDANS, were subjected to amino acid sequencing. The NH2-terminal amino acid sequences of those peptides are shown along with the deduced amino acid sequence (Figure 5 ) . The unique fluorescent peptide, C9 1V-3, contained an NH,-terminal amino acid sequence exactly the same as the deduced sequence from residues 2 (Glf4) to 13 (Leu95)except for residue 9 (Cys9'), which was replaced with a valine residue as expected from the muta-
Biochemistry, Vol. 30, No. 18, 1991 4591
Bacillus polymyxa 8-Amylase
Table I: Kinetic Parameters of Various &Amylases with Soluble Starch as a Substrate'
m c
14.5 f 0.1 31.5 C83S 0.460 f 0.007 0.099 f 0.021 9.5 f 0.1 C91V 96.6 0.118 f 0.012 40.5 f 4.0 C323S 343 0.549 f 0.005 16.2 f 0.2 29.5 C-free 'Initial reaction rates were determined at 25 OC as described in Materials and Methods. Kinetic data were fitted to the MichaelisMenten relationship to evaluate K, and Vmx. Turnover number (kaJ was calculated on the basis of the amount of enzyme used.
0
N N
c, 0)
0
c n
k
n
